The control of androgen production by the Leydig cell is directly regulated by luteinizing hormone via specific receptors for LH/hCG. In vivo treatment with endogenous or exogenous gonadotropin causes initial LH receptor up-regulation followed by down-regulation. Large doses of hCG cause "early" (prior to pregnenolone) and "late" (17 -hydroxylase, 17-20 desmolase) lesions that are independent of receptor loss or protein kinase activation. Such negative control by LH of receptors and responses is not observed in immature or fetal Leydig cells in vivo or in vitro, where only up-regulation was demonstrated. The goal of this project is to understand the steps involved in the hormonal control of steroidogenesis. We have extended the studies and have developed rapid and convenient methods using combined centrifugal elutriation and metrizamide gradients for complete purification of large quantities of functionally intact Leydig cells, to facilitate analysis of the mechanisms of gonadotropin action in vitro. The Leydig cell population is composed of cells with different densities and sedimentation velocities but with similar morphology, biological activity, and susceptibility to desensitization by gonadotropins. The latter caused considerable reduction of cellular density, possibly related to changes in lipid content. Exposure of cultured adult Leydig cells to chemically deglycosylated hCG caused no receptor down-regulation or steroidogenic lesions due to the predominant antagonist activity of this derivative. We have also shown that the fetal rat Leydig cell can be maintained in culture with retention of their LH-mediated steroidogenic responses and with expression of functional GnRH receptors. The demonstrated inhibitory actions of GnRH on steroidogenesis, with expression of GnRH receptors in the fetal and post-natal testes, are in contrast to the up-regulatory functions of the microsomal enzymes by gonadotropins, and indicate that GnRH could influence the actions of gonadotropins upon Leydig cell function in the neonatal testis. We will proceed with studies on the biochemical definition of hormone-induced steroidogenesis in fetal and adult Leydig cell.